Background/Purpose: The synovial lymphatic system (SLS) maintains joint homeostasis. Post-traumatic osteoarthritis (PTOA) has increased lymphatic vessels (LVs), but decreased SLS drainage. LVs are routinely studied by immunostaining of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE1) on tissue sections, but it cannot address the discrepancy between increased LV #s and decreased SLS function in PTOA joints. Lightsheet microscopy allows observation of the full depth of a 600-700mm mouse joint, compared to 4-10mm in 2D imaging. Here we describe the first lightsheet microscopy study of healthy vs. PTOA mouse knees for distribution and alteration of synovial LVs. We hypothesize that lightsheet 3D imaging is superior to traditional immunohistochemistry for comprehensive LV network analyses of mouse knee joints in full depth, which provides new information of SLS.

Methods: Knee joints of 5-m-old C57 male mice receiving sham or PTOA surgery (n=3/group) were stained for synovial LVs with anti-LYVE1. Samples were subjected to lightsheet microscopy and a total of 550-650 consecutive 1mm slices were collected for volumetric/branching analysis in Imaris 10.2 software.

Results: 1. 3D reconstruction of LVs reveals extensive LV network compared to a 2D image in the normal joint (Fig.1). One 1μm thick image of lightsheet 2D data resembled a typical LYVE1 immunohistochemistry of the knee, where the LYVE1+ signals were located in the synovium on the patella side, forming long unbranched vessel-like structures. Few LYVE1+ signals were on the popliteal side (1A). 3D reconstruction of 622μm in depth revealed LYVE1+ signals on both side of the joint, forming extensive LV structure with complex branching (1B). To visualize the LV branching structure, the 3D reconstructed image was rotated towards the patella (1B middle) and popliteal side (1B right). Notably, the star shows 2 circular LYVE1+ structures at the tibial growth plate level in the 2D (1A) were 2 LVs along the tibial axis in 3D (1B middle). 2. Synovial LVs in PTOA joints have increased branches with shorter length, thinner diameter, and smaller volume (Fig.2). We performed quantitative branching analysis, recognizing LV structures in grey lines, and branching points in red balls. PTOA joints had increased branching that were smaller and shorter (2A-B): increased branch # (197±22 vs. 74±9 in sham, p=0.0008) (2C), decreased mean branch length (59±7 vs. 146±3μm in sham, p< 0.0001) (2D), diameter (15±5 vs. 29±5μm in sham, p=0.03) (2E), and smaller LVYE1+ volume (0.36±0.11 vs. 12±4% in sham, p=0.03) (2G).

Conclusion: PTOA induces local lymphangiogenesis with impaired function. We found decreased LV volume (3D) despite increased LV branching #s (2D) in PTOA joints. Possible explanations for the difference between 2D and 3D analysis are: 1) 2D area underestimates the diameter changes; and 2) histology sections (4-10μm) underrepresents the fewer LV branches with ~30μm-diameter. Thus, impaired SLS draining function in PTOA joints are likely due to decreased LV volume. In conclusion, lightsheet imaging/3D analysis is an advantaged tool for studying SLS than the conventional immunostaining, allowing the observation of LV network in full depth of joints.

Fig 1. 3D reconstruction of the synovial lymphatic vessels from light sheet images reveals much more extensive lymphatic network than those from a 2D image. Knee joints of 5-m-old C57Bl/6 male mice were processed for the lightsheet microscopy. The synovial LVs were labeled with anti-LYVE1/AlexaFluor647 antibody. Joints were mounted in a sagittal position, and a series of 550-650 consecutive images (=slices) were collected from the medial side of the joint with a step size of 1mm. Fluorescence signal were collected in the AlexaFluor647 channel to visualize the LYVE1+ LVs (green) and GPF channel to visualize the anatomic structure of the joint using the GPF autofluorescence of the bone. (A) A 2D cross-section of 1 mm from the series of 622 slices collected for this joint. The autofluorescence signal shows the histology landmarks, including the distal end of femur and the proximal end of tibia with articular cartilage, subchondral bone, growth plates, and menisci (M). Arrow: most LYVE1+ signals are located on the anterior (patellar) side as unbranched vessel-like structures. Arrowheads: a possible lymphatic vessel branch point. Star: two circular LYVE1+ structures near the tibial growth plate. Note that since this is a 2D cross-section, whether they are two branching vessels perpendicular to the observation plane or two vessels parallel to the tibia axis is unclear. (B) 3D reconstruction of the synovial LVs of the joint (622mm), including sagittal, patella, and popliteal views. Anatomical structures of the joint can be observed. Note that in contrast to the 2D image, both the patella side and popliteal side have initial LVs with extensive branching. *Note in the patella rotation view, at the level of the tibial growth plates, two LVs are parallel to the tibial axis, which as represented as the two circular structures in the 2D cross-section image. Scale bar= 500mm

Fig 2. Synovial lymphatic vessels in PTOA joints have increased branches, but shorter length, thinner diameter and smaller volume. 3D reconstruction showing LYVE1+ LVs (green) in the sham and PTOA joints. Branch analysis was performed using the “filament” module in Imaris10.2 (Oxford), which identifies branches (grey) and branch points (red). (A&B) Initial LVs in the PTOA joint visibly differs from sham. (C) Branch number/joint. (D) The mean length of all individual branches recognized by Amira in one joint. (E) The mean diameter of all individual branches recognized by Amira in one joint. (F) The total volume of LYVE1+signal of one joint. (G) LYVE1+LV volume/the volume of the synovial tissues. Student t-test were used to assess the statistical difference between sham and PTOA group.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/lightsheet-imaging-with-3d-analysis-for-wholistic-assessment-of-synovial-lymphatic-vasculature-within-joints-of-murine-models-of-arthritis/